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Abstract

Objective— An excess of metalloproteinases (MMPs) over tissue inhibitors of metalloproteinases (TIMPs) may favor atherosclerotic plaque rupture. We compared TIMP levels in nonfoamy and foam-cell macrophages (FCM) generated in vivo.

Methods and Results— In vivo generated rabbit FCM exhibited 84% reduced TIMP-3 protein compared to nonfoamy macrophages, and immunocytochemistry revealed a TIMP-3 negative subset (28%). Strikingly, only TIMP-3 negative FCM invaded a synthetic basement membrane, and invasion was inhibited by exogenous TIMP-3. TIMP-3 negative FCM also had increased proliferation and apoptosis rates compared to TIMP-3 positive cells, which were retarded by exogenous TIMP-3; this also reduced gelatinolytic activity. TIMP-3 negative FCM were found at the base of advanced rabbit plaques and in the rupture-prone shoulders of human plaques. To explain the actions of low TIMP-3 we observed a 26-fold increase in MT1-MMP (MMP-14) protein in FCM. Adding an MT1-MMP neutralizing antibody reduced foam-cell invasion, apoptosis, and gelatinolytic activity. Furthermore, MT1-MMP overexpressing and TIMP-3 negative FCM were found at the same locations in atherosclerotic plaques.

Conclusions— These results demonstrate that TIMP-3 is downregulated in a distinct subpopulation of FCM which have increased MMP-14. These cells are highly invasive and have increased proliferation and apoptosis, all properties expected to destabilise atherosclerotic plaques.

Macrophages have been proposed to be involved in both atherosclerotic plaque fibrous cap formation and disruption.1–4 Matrix metalloproteinases (MMPs) are one group of proteases produced by macrophages that also appear to have a dual role in plaque cap building and destruction. Consistent with this, MMPs-2 and -9 in particular are implicated in intima formation after vascular injury. On the other hand, MMPs-1, -2, -3, -7, -9, -11, -12, -13, -14, and -16 are upregulated in human plaques in macrophage-rich areas that show a high propensity for plaque rupture.5 Numerous MMPs are also upregulated in the plaques of cholesterol-fed rabbits6,7 and ApoE null mice.8 Studies with ApoE/MMP compound knockout mice and other transgenic models show clear effects of individual MMPs on both fibrous cap formation and rupture.5 What seems to determine the outcome is the level and spectrum of MMPs produced and activated. There is therefore significant interest in the factors regulating MMP activity in macrophages. Conversion of macrophages to foam-cells is one important factor. For example, in vivo generated rabbit foam-cells have increased levels of MMPs-1 to 3 and -12.6,9,10

Tissue inhibitors of MMPs (TIMPs) are a family of specific protein inhibitors of MMPs, 4 of which have been demonstrated in vascular cells. Smooth muscle cells (SMCs) in human atherosclerotic plaques harbor abundant TIMP-1 and -2 protein expression.11 TIMP-1 and -2 expression is increased at the base of atherosclerotic plaques in cholesterol-fed rabbit aortas, and this correlates with areas of low MMP activity measured by in situ zymography.12 TIMP-3 has been detected more selectively in plaque macrophages at the shoulder regions and between the fibrous cap and necrotic core.13 Subsequently it was suggested that TIMP-3 may serve as a protective mechanism against plaque rupture by dampening local proteolysis.13 Thus, upregulation of TIMPs could have an important plaque stabilizing effect. Consistent with this, gene transfer of TIMP-1 or -2 reduces atherosclerosis and stabilizes plaques in the apoE knockout mouse.14,15 The effect of foam-cell formation on TIMP production has not been systematically investigated. We therefore undertook a study to compare the expression of TIMPs during macrophage foam cell formation.

Methods

Please see expanded details in the Online Data Supplement (available online at http://atvb.ahajournals.org).

Generation and Isolation of Macrophages and Foam-Cell Macrophages

New Zealand White rabbits (Harlan, UK) fed a normal chow diet or 1% cholesterol-supplemented diet had sterile sponges placed under the dorsal skin to generate nonfoamy macrophages (NFMs) or foam-cell macrophages (FCMs), respectively, as described previously.10 Endarterectomy specimens were made available from the AtheroExpress biobank.16

Total RNA was extracted from rabbit macrophage and foam-cell macrophages. Procedures for RT-PCR have been described previously.9 Results are expressed in arbitrary units and are adjusted for 18S mRNA levels.

Western Blotting

Cell lysates and concentrated supernatants (10×) from both human and rabbit NFM and FCM were subjected to Western blotting and TIMP-3 or MT1-MMP levels determined.

In Vitro Studies on Macrophages and Macrophage-Derived Foam Cells

The effect of recombinant TIMP-3 (10 nmol/L) on migration, proliferation, and apoptosis were determined in rabbit NFM and FCM as previously described.15 Additionally parallel studies using a MT1-MMP neutralizing antibody (10 nmol/L) were performed. To induce apoptosis, rabbit NFM and FCM were either cultured in the presence of lipopolysaccharide (LPS) or subjected to serum withdrawal for 72 hours, with or without the addition of either recombinant TIMP-3 (10 nmol/L) or a MT1-MMP neutralizing antibody (10 nmol/L).

In Situ Zymography

Rabbit NFM and FCM were cultured on glass coverslips and incubated in the presence of fluorescently quenched DQ gelatin substrate (40 μg/mL). Parallel experiments with either recombinant TIMP-3 (10 nmol/L) or a MT1-MMP neutralizing antibody (10 nmol/L) were performed. Gelatinolytic activity was visualized under fluorescent microscopy.

Immunohistochemistry

Immunohistochemistry was conducted for macrophages, TIMP-3, MT1-MMP, and cleaved caspase-3 (apotosis). Dual immunostaining was performed using the same antibodies and visualized by confocal microscopy.

Statistical Analysis

Data were analyzed for normality and compared using the unpaired Student t test or the Mann–Whitney test, as appropriate. Statistical differences between apoptotic foam-cell macrophages were analyzed by Student paired t test. Normally distributed data are presented as mean±SEM.

Results

TIMP-3 mRNA and Protein Expression of Nonfoamy and Foam-Cell Macrophages

RAM-11 immunocytochemistry stained 98% of all rabbit FCM and NFM, as previously demonstrated.10 TIMP-3 immunocytochemistry revealed that all rabbit NFM expressed TIMP-3 protein at similar levels (Figure 1A). By contrast FCM showed a wide spectrum of TIMP-3 protein; indeed, a subset (28±5%) was TIMP-3 negative (arrows in Figure 1B). When the invasion of rabbit FCM through a synthetic basement membrane was tested in vitro, remarkably 100% of the macrophages that penetrated were TIMP-3 negative (Figure 1D and 1E) and these cells spread out to adopt an elongated morphology. By contrast, 100% of the cells that did not penetrate after 48 hours were TIMP-3 positive, failed to spread, and kept a rounded morphology (Figure 1D and 1E). These results were reproduced in 5 separate experiments. Immunocytochemistry for RAM11 confirmed that all the cells that had invaded were macrophages (Figure 1F). Immunocytochemistry for TIMP-1 and TIMP-2 showed no difference between migrated and nonmigrated FCM (results not shown). Addition of recombinant TIMP-3 to the Matrigel significantly reduced the invasion of FCM (65%, P<0.05; supplemental Table I) and the nonmigrated cells now all failed to spread and were a mixture of TIMP-3 positive and negative cells (Figure 1C). TIMP-3 addition had no effect on the invasion of NFM (supplemental Table I).

Localization of TIMP-3 Protein in Rabbit and Human Atherosclerotic Plaques

We sought histological evidence in plaques to corroborate our in vitro findings of variable TIMP-3 expression in FCM and its relation with invasion. The early fatty streak lesions formed after feeding rabbits cholesterol for 4 weeks demonstrated areas rich in macrophages and FCM (Figure 4A), which colocalized with cell-specific TIMP-3 protein expression (Figure 4B). Whereas most NFM were strongly TIMP-3 positive (arrows, Figure 4C), a more divergent expression pattern was observed within FCM, throughout the lesion (Figure 4C). Additionally, extracellular TIMP-3 protein was detected at the lesion/media interface (Figure 4C). Based on the use of nonimmune mouse IgG as a primary antibody, TIMP-3 staining was highly specific (Figure 4D). In advanced plaques formed after 8 weeks of cholesterol feeding, FCM within the upper aspect of the lesions (Figure 4E) demonstrated TIMP-3 immunoreactivity (arrows, Figure 4F). However, the FCM found in the deeper layers of the plaque (Figure 4E) showed little or no TIMP-3 protein expression (Figure 4G). Interestingly, dual immunohistochemistry revealed that the NFM in the media below the lesion (presumably resident macrophages) are TIMP-3 positive (arrowheads; Figure 4H). Also, adventitial macrophages underlying the lesion are clearly nonfoamy and express abundant TIMP-3 (arrows; Figure 4H). TIMP-1 and TIMP-2 expression was observed in NFM and FCM throughout the lesion (data not shown).

MT1-MMP mRNA and Protein Expression of Nonfoamy and Foam-Cell Macrophages

Considering the effects observed with TIMP-3 and that we have previously observed similar in vitro effects on migration, proliferation, and apoptosis with TIMP-2 but not TIMP-1, we investigated the expression patterns of MT1-MMP, which is inhibited by TIMP-2 and TIMP-3 but not TIMP-1.17 Both MT1-MMP mRNA (2.6-fold; P=0.0138; supplemental Figure IC) and protein (26-fold; P<0.0001; supplemental Figure IA and IB) were significantly elevated in FCM compared to NFM control macrophages.

Only 23% of the rabbit FCM that failed to penetrate the synthetic basement membrane were MT1-MMP positive (Figure 3B), whereas 100% of the cells were TIMP-3 positive (Figure 1C). Conversely 100% of the FCM that invaded the membrane were MT1-MMP positive (Figure 3C), whereas none were TIMP-3 positive (Figure 1E). This implies that lack of TIMP-3 combined with upregulated MT1-MMP (and possibly other MT-MMPs) in these FCM permits them to invade. Consistent with this, proteolytic activity was reduced in FCM by adding an MT1-MMP neutralizing antibody (60%; P<0.01; Figure 3A). Addition of MT1-MMP neutralizing antibody to the Matrigel also tended to reduce the invasion of FCM (71%, P=0.09). Furthermore, the rate of apoptosis in foam-cell macrophages was significantly inhibited by a MT1-MMP neutralizing antibody (6±2 versus 19±9%, P<0.05; Figure 2A).

We went on to use immunocytochemistry to relate our findings with isolated cells to human and rabbit plaques. MT1-MMP positive FCM were found at the shoulder regions of human atherosclerotic plaques. Moreover, the frequency of such cells undergoing apoptosis was significantly higher than observed for TIMP-3 positive FCM (27±9% v 4±1%, P<0.05, Figure 5J). MT1-MMP demonstrated a converse pattern of localization in advanced rabbit atherosclerotic plaques compared to TIMP-3 (Figure 6). The majority of macrophages (Figure 6A and 6D) within the upper aspect of the lesion were MT1-MMP negative, whereas FCM within the deepest region of the plaque were MT1-MMP positive (Figure 6B and 6E). MT1-MMP positive foam-cells were TIMP-3 negative (Figure 6C and 6F).

Discussion

It was previously shown that the phenotypic transformation of monocytes to macrophages in culture coincides with upregulation of TIMP-3 mRNA and protein.13 Consistent with this, TIMP-3 protein expression was demonstrated in macrophages in human atherosclerotic plaques and was suggested to act as a protective factor against plaque rupture.13 Our studies illustrate, for the first time, that transformation of differentiated macrophages to FCM causes a subsequent decrease in TIMP-3 protein expression, and that the protective effect of TIMP-3 is completely lost in a subpopulation of FCM that also overexpresses MMP-14. We observed no difference in TIMP-3 mRNA expression between NFM and FCM, consistent with previous observations that posttranscriptional mechanisms can regulate TIMP expression during macrophage development.18

Our most interesting and unexpected finding was that only the subset of FCM that became TIMP-3 negative was capable of invading through a synthetic extracellular matrix. Furthermore, TIMP-3 negative FCM had an increased propensity to undergo proliferation and apoptosis. It was important to determine whether the results obtained in FCM and NFM derived from granulomas are relevant to plaque macrophages. To do this we showed that FCM with high and low TIMP-3 expression are found in early fatty streak lesions in rabbit aortas, whereas in the deep layers of advanced atherosclerotic plaques, most FCM had little or no TIMP-3 protein expression. The fact that TIMP-3 negative macrophages occurred in the deeper layers of the plaque is consistent with their greater invasive ability. Interestingly, we recently demonstrated upregulation of MMP-12 and downregulation of arginase-I occurs in the same location as TIMP-3 down regulation in advanced rabbit plaques.10

It is possible that TIMP-3 downregulation marks a distinct phenotype of macrophages. Functionally divergent macrophage subtypes have been ascribed to other inflammation-related pathologies19,20 and recently demonstrated to populate murine21,22 and human atherosclerotic lesions.23,24 Further studies beyond the scope of the present experiments would be needed to establish the relationship between the TIMP-3 negative subpopulation described here and these previously described phenotypes.

We also identified wide variation in TIMP-3 levels in FCM in human plaques. Looking specifically at the shoulder-regions of advanced plaques, islands/nodules of FCM with either low or no TIMP-3 expression were apparent, surrounded by TIMP-3 positive FCM. These areas were predominantly around the periphery of the lipid/necrotic core, below the thinnest regions of the fibrous cap. Although it was not commented on, the immunocytochemical data previously reported by Fabunmi and colleagues also shows a subpopulation of macrophages that appears TIMP-3 negative at similar regions.13

The most obvious explanation for the effect of TIMP-3 deficiency in FCM is that it reveals the actions of metalloproteinases that facilitate invasion and proliferation and induce susceptibility to apoptosis. Consistent with this explanation, exogenous TIMP-3 blocked reduced invasion, proliferation, and apoptosis of FCM. Despite reported findings that TIMP-3 is proapoptotic in some cells,25,26 prosurvival effects have also been described in other cell types.27–29 We recently demonstrated that TIMP-2 similarly reduces FCM migration, proliferation, and apoptosis, although exogenous TIMP-1 has no effect.15 These findings suggest the involvement of a membrane type MMP. MT1-MMP (MMP-14), for example, is inhibited by TIMP-2 and -3 but very poorly by TIMP-1.17 Furthermore, MMP-14 is upregulated during foam cell formation in vitro.30 We demonstrated here that mRNA and protein are elevated in in vivo generated FCM compared to NFM. Additionally, it has recently been observed that MMP-14 regulates intercellular adhesion molecule-1 (ICAM-1) mediated monocyte/macrophage migration through an endothelial monolayer in vitro; TIMPs-2 and -3 blocked migration, whereas TIMP-1 was ineffective.31 We now illustrate that blocking MMP-14 activity retards FCM invasion, diminishes their proteolytic potential, and reduces their susceptibility to undergo apoptosis, similar to that observed with exogenous TIMP-3. Supportive findings have shown that MMP-14 can induce apoptosis in several cell types.32,33 Thus we propose that transformation of macrophages to FCM causes a significant decrease in TIMP-3 protein while increasing MMP-14 levels. This inverse correlation implies that the protective effect of TIMP-3 is reduced and the disruptive nature of MMP-14 is heightened. Our studies cannot rule out that other MT-MMPs also contribute to these effects, because several are also inhibited by TIMP-2 and -3 but not TIMP-1.

Consistent with our data, a previous study also found prominent MMP-14 expression in FCM at the base of advanced rabbit lesions.34 It is interesting that the subpopulation of FCM that have diminished TIMP-3 expression reside in a similar area to where increased MMP-14 (Figure 6) is detected in both the rabbit and human advanced plaques.10 MMP-14 expression has been postulated to accelerate the progression of atherosclerotic plaques and promote plaque instability.35,36 Moreover, MMP-14 has been shown to be fundamental for macrophage-mediated proteolysis and invasion.37 Thus the reduction in TIMP-3 and increased MMP-14 expression suggests that a more invasive and destructive subpopulation of FCM resides at the base of rabbit and the shoulder region of human plaques.

In summary our results show that TIMP-3 is downregulated in a subset of FCM, which results in increased invasion through ECM, proliferation, and apoptosis. Additionally, the loss of TIMP-3 appears associated with an increase in MMP-14 expression and activity. The ability of FCM to degrade ECM has been previously associated with morphological features of plaque instability.6,11 Increased foam-cell proliferation and apoptosis, resulting in increased extracellular lipid content, are also associated with plaque instability.38 Hence all these properties promoted by reducing the TIMP-3/MMP-14 balance would be expected to increase the potential of FCM to destabilise atherosclerotic plaques. The identification of FCM with differing proteolytic potential may aid future development of therapies to promote atherosclerotic plaque stability.